Recombinant Non-Animal Cell for Making Biliverdin

ABSTRACT

Methods for producing biliverdin in a microorganism, methods for producing biliverdin from a non-animal source, cells for producing biliverdin and methods for producing cells for producing biliverdin are disclosed.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/854,791, filed Apr. 1, 2013, which is adivisional application of U.S. patent application Ser. No. 12/939,880,filed Nov. 4, 2010, which claims priority to and the benefit of U.S.Provisional Patent Application No. 61/258,126, filed Nov. 4, 2009, eachof which are hereby incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The attached paper copy of the Sequence Listing is submitted incompliance with 37 C.F.R. §§1.821-1.825 and is incorporated by referenceherein. A computer-readable copy of the sequence listing may be found inU.S. patent application Ser. No. 12/939,880, the entirety of which isincorporated by reference in its entirety. Applicants request the use ofthis computer-readable sequence listing. The contents of the paper copy(filed herewith) contain no new matter and the paper copy andcomputer-readable copy of the Sequence Listing are identical.

BACKGROUND

Biliverdin IX-alpha (biliverdin IXα) is the most common form of severalbiliverdin isomers found in nature (FIG. 1C). It is produced from heme(also named iron protoporphyrin IX) (FIG. 1A) by the enzyme hemeoxygenase. In animals, biliverdin IXα is reduced by the enzymebiliverdin reductase to bilirubin IXα which is the major form of severalbilirubin isomers (FIG. 1B). One known role of biliverdin IXα in natureis in animals as an intermediate in hemoglobin breakdown as red bloodcells are degraded in phagocytes (FIG. 2). The hemoglobin prostheticgroup, heme, with its bound iron, is released in this degradativeprocess, and heme is converted by HO to biliverdin IXα. Biliverdin IXαis then converted to bilirubin IXα by the enzyme biliverdin reductase(FIG. 2). Bilirubin IXα is consecutively bound to serum albumin and thenin the liver to glucoronic acid (conjugated bilirubin) which confers arelatively high degree of water solubility. Conjugated bilirubin is thenexcreted in the bile. Overall, this process is viewed as a process foranimals (e.g. humans) to degrade and eliminate heme—which is toxic whenaccumulated.

Biliverdin IXα is also made by microbes. For example, biliverdin IXα isa precursor to microbial phycobilins, i.e. phycocyanobilin (pcb) andphycoerythrobilin (peb). Pcb and peb are the pigment molecules for thelight-harvesting complexes of photosynthetic cyanobacteria, phycocyaninand phycoerythrin, respectively. These complexes collect light energy(for example solar energy) and funnel it to photosynthetic reactioncenters where the energy is converted into chemical energy) (FIG. 3).Pcb is also the pigment for phytochrome—a light sensing receptor thatoccurs in plants and other cells. An analogous receptor,bacteriophytochrome, is found in certain bacteria. The pigment forbacteriophytochrome is biliverdin IXα rather than pcb, which reveals yetanother biological role for biliverdin IXα. These latter bacteria—likeall microbes—either do not produce or do not accumulate bilirubin IXα.The lack of bilirubin IXα accumulation by microbes is either aconsequence of lacking biliverdin reductase or the conversion ofbilirubin IXα to bile pigments such as those involved in photosyntheticlight-harvesting.

Bilirubin IXα is known to associate with cell membranes where itquenches the propagation of reactive oxygen species (ROS). It istherefore believed to confer protection to membrane lipid and proteincomponents against oxidative damage. Thus, an additional suggestedfunction of biliverdin IXα is to serve as the immediate source ofbilirubin IXα which in turn acts as a cytoprotective antioxidant andanti-inflammatory agent against cell damaging ROS (FIG. 4). Althoughbilirubin IXα (and not biliverdin IXα) is believed to be thecytoprotective antioxidant, it is observed that biliverdin IXα is moreeffective than bilirubin IXα when administered at tissueinjury/inflammatory sites where ROS are prevalent. One explanation forthe higher efficacy of biliverdin IXα is that it is more hydrophilicthan bilirubin IXα and therefore has better access to tissue sites whereit is then reduced by biliverdin reductase to bilirubin IXα. Anotherexplanation is that when biliverdin IXα binds to biliverdin reductase,this enzyme is activated and initiates a cell signaling cascade thatresults in the production of the anti-inflammatory cytokineinterferon-10.

There is increasing evidence that biliverdin IXα can be used as acytoprotective therapeutic agent. Examples of clinical applications ofbiliverdin IXα include treatment of vasoconstriction (U.S. PatentApplication Publication No. 20030027124); coronary artery disease(artherosclerosis); ischemia/reperfusion injuries after smallintestinal, heart, and kidney transplantation; severe sepsis; injuriesfrom liver grafts; and prevention of intimal hyperplasia induced byvascular injury. Today, biliverdin IXα is predominantly derived bychemical oxidation of bilirubin IXα or by using the enzyme bilirubinoxidase (U.S. Pat. No. 5,624,811). Bilirubin IXα is extracted from thebile of various mammals, especially from swine or other livestock.Commercial animal bilirubin IXα preparations are often contaminated withconjugated bilirubin and isomers (e.g. bilirubin XIIIα) (Reisinger etal. 2001; U.S. Pat. No. 431,166). As a result, biliverdin IXα derivedfrom bilirubin IXα preparations using oxidative processes or enzymes mayalso contain isomers. The clinical consequences of using biliverdin IXαcontaminated with such isomers are not clear. In addition, the use ofbiliverdin IXα preparations derived from animal bilirubin carries therisk of prion contamination often associated with materials derived fromanimal sources.

A recent claim (U.S. Pat. No. 7,504,243) for biliverdin IXα productionby a yeast depends on addition of hemoglobin (from animal blood) to thegrowth culture as a source of heme. Another report shows biliverdin IXαsynthesis by Escherichia coli expressing a heterologous HO gene ofanimal origin (rat). The biliverdin IXα was produced at low levels andappears to remain cell-bound.

The limited amounts of biliverdin IXα produced by yeast and E. coliexpressing heterologous HO genes could result from restricted access toheme. In E. coli, the biosynthesis of heme is regulated at the initialstep of tetrapyrrole biosynthesis—the synthesis of 5-aminolevulinic acid(ALA) by the C5-pathway. The C5 pathway involves conversion of glutamateto glutamyl-tRNA by glutamyl-tRNA synthetase, reduction toglutamate-γ-semialdehyde by an NADPH-dependent glutamyl tRNA reductaseand transamination by glutamate-γ-semialdehyde aminomutase to form ALA.The C5 pathway is feedback-inhibited by heme and, as a consequence, thecellular levels of heme are kept low. In contrast, mammals, plants, andcertain bacteria such as Rhodobacter sphaeroides produce ALA fromglycine and succinyl-CoA via the enzyme ALA synthetase. This lattermechanism for ALA biosynthesis is termed the “04 pathway.” The C4pathway ALA synthetase is not subject to feedback inhibition by heme. Ittherefore allows the accumulation of heme and higher cellularconcentrations. When combined with the C4 pathway for ALA synthesis, HOwill have greater access to its substrate, heme, resulting in increasedpotential for producing biliverdin IXα.

SUMMARY

Methods for producing biliverdin in a microorganism, methods forproducing biliverdin from a non-animal source, cells for producingbiliverdin and methods for producing cells for producing biliverdin aredisclosed. These methods and cells conform to a general strategy forenhanced production of biliverdin for a non animal source. This generalstrategy is depicted in FIG. 14.

In one aspect, a method of producing biliverdin in a microorganism isdisclosed, comprising: culturing a cell comprising a recombinant hemeoxygenase and a recombinant heme biosynthetic enzyme.

In certain embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme oxygenase. In certainother embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme biosynthetic enzyme.The cell may also comprise a regulatable promoter operably linked to apolynucleotide which codes for the heme oxygenase. In a relatedembodiment, the cell comprises a regulatable promoter operably linked toa polynucleotide which codes for the heme biosynthetic enzyme. Theregulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase.In a related embodiment, the heme biosynthetic enzyme is hemA. Inanother related embodiment, the heme biosynthetic enzyme is an ALAsynthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme.In a related embodiment, the heme oxygenase enzyme is HO1. In anotherrelated embodiment, the heme oxygenase enzyme is an HO family analog.

In certain other embodiments, lactose is provided to the cell. Lactosemay be provided in an initial concentration of from about 2% (w/v) toabout 10% (w/v). Furthermore, the methods may further comprise the stepof providing a trace metal to the cell. Trace metals may be added aspart of a trace metal solution. Some or all of NaCl, ZnSO₄, MnCl₂,FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine or CaCl₂ may beprovided to the cell.

In certain other embodiments, a plurality of measurements of thedissolved oxygen concentration in the growth medium of the cell may betaken. In a related set of embodiments, at least an initial measurement,a first intermediate measurement, a second intermediate measurement, anda third intermediate measurement of dissolved oxygen concentration inthe growth medium of the cell may be taken. In related embodiments, theinitial measurement yields an initial value, the first intermediatemeasurement yields a first intermediate value less than the initialvalue, the second intermediate measurement yields a second intermediatevalue greater than the first intermediate value, and the thirdintermediate measurement yields a third intermediate value less than thefirst intermediate value. In further related embodiments, dissolvedoxygen in the growth medium in concentrations at the initial value, thefirst intermediate value, the second intermediate value and the thirdintermediate value indicates that biliverdin will be produced.

In certain other embodiments, the biliverdin is excreted into the growthmedium of the cell. In one such embodiment, foam accumulates in thegrowth medium of the cell and a portion of the biliverdin excreted in tothe growth medium of the cell is located with the foam. In relatedembodiments, the foam is collected. In any embodiment, the biliverdinmay be collected.

In another aspect, a method of producing biliverdin from a non-animalsource is disclosed, comprising: culturing a cell comprising arecombinant heme oxygenase and a recombinant heme biosynthetic enzyme.

In certain embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme oxygenase. In certainother embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme biosynthetic enzyme.The cell may also comprise a regulatable promoter operably linked to apolynucleotide which codes for the heme oxygenase. In a relatedembodiment, the cell comprises a regulatable promoter operably linked toa polynucleotide which codes for the heme biosynthetic enzyme. Theregulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase.In a related embodiment, the heme biosynthetic enzyme is hemA. Inanother related embodiment, the heme biosynthetic enzyme is an ALAsynthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme.In a related embodiment, the heme oxygenase enzyme is HO1. In anotherrelated embodiment, the heme oxygenase enzyme is an HO family analog.

In certain other embodiments, lactose is provided to the cell. Lactosemay be provided in an initial concentration of from about 2% (w/v) toabout 10% (w/v). Furthermore, the methods may further comprise the stepof providing a trace metal to the cell. Trace metals may be added aspart of a trace metal solution. Some or all of NaCl, ZnSO₄, MnCl₂,FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO4, thiamine or CaCl2 may beprovided to the cell.

In certain other embodiments, a plurality of measurements of thedissolved oxygen concentration in the growth medium of the cell may betaken. In a related set of embodiments, at least an initial measurement,a first intermediate measurement, a second intermediate measurement, anda third intermediate measurement of dissolved oxygen concentration inthe growth medium of the cell may be taken. In related embodiments, theinitial measurement yields an initial value, the first intermediatemeasurement yields a first intermediate value less than the initialvalue, the second intermediate measurement yields a second intermediatevalue greater than the first intermediate value, and the thirdintermediate measurement yields a third intermediate value less than thefirst intermediate value. In further related embodiments, dissolvedoxygen in the growth medium in concentrations at the initial value, thefirst intermediate value, the second intermediate value and the thirdintermediate value indicates that biliverdin will be produced.

In certain other embodiments, the biliverdin is excreted into the growthmedium of the cell. In one such embodiment, foam accumulates in thegrowth medium of the cell and a portion of the biliverdin excreted in tothe growth medium of the cell is located with the foam. In relatedembodiments, the foam is collected. In any embodiment, the biliverdinmay be collected.

In another aspect, a cell comprising a recombinant heme oxygenase and arecombinant heme biosynthetic enzyme is disclosed.

In certain embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme oxygenase. In certainother embodiments, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme biosynthetic enzyme.The cell may also comprise a regulatable promoter operably linked to apolynucleotide which codes for the heme oxygenase. In a relatedembodiment, the cell comprises a regulatable promoter operably linked toa polynucleotide which codes for the heme biosynthetic enzyme. Theregulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase.In a related embodiment, the heme biosynthetic enzyme is hemA. Inanother related embodiment, the heme biosynthetic enzyme is an ALAsynthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme.In a related embodiment, the heme oxygenase enzyme is HO1. In anotherrelated embodiment, the heme oxygenase enzyme is an HO family analog.

In another aspect, methods of producing a cell for producing biliverdinare disclosed. In certain embodiments, a polynucleotide comprising asequence which codes for a recombinant heme oxygenase is introduced intoa parent cell comprising a recombinant heme biosynthetic enzyme. Incertain other embodiments, a polynucleotide comprising a sequence whichcodes for a recombinant heme biosynthetic enzyme is introduced into aparent cell comprising a recombinant heme oxygenase. In certain otherembodiments, the method comprises introducing into a parent cell a firstpolynucleotide comprising a first sequence which codes for a recombinantheme oxygenase; and a second polynucleotide comprising a second sequencewhich codes for a recombinant heme biosynthetic enzyme.

In other embodiments, a polynucleotide comprising a first sequence whichcodes for a recombinant heme oxygenase and a second sequence which codesfor a recombinant heme biosynthetic enzyme is introduced into a parentcell.

In certain other embodiments, a first polynucleotide comprising apromoter sequence is introduced into a parent cell, and the firstpolynucleotide recombines with a second polynucleotide comprising asequence which codes for a heme oxygenase, such that the firstpolynucleotide and a portion of the second polynucleotide form a linkedpolynucleotide which codes for a recombinant heme oxygenase.

In another set of embodiments, a first polynucleotide comprising apromoter sequence is introduced into a parent cell, and the firstpolynucleotide recombines with a second polynucleotide comprising asequence which codes for a heme biosynthetic enzyme, such that the firstpolynucleotide and a portion of the second polynucleotide form a linkedpolynucleotide which codes for a recombinant heme biosynthetic enzyme.

For any method of producing a cell for producing biliverdin, introducinga polynucleotide into a cell may be performed by transformation. Thecell used may be a microorganism, bacterial cell, or an Escherichia colicell. Furthermore, the methods of producing biliverdin may compriseculturing cells comprising a polynucleotide which has been optimized forexpression in a cell. The cells for producing biliverdin may compriseculturing cells comprising a polynucleotide which has been optimized forexpression in a cell. Furthermore, the methods of producing cells forproducing biliverdin may comprise introducing a polynucleotide into theparent cell, the polynucleotide having been optimized for expression ina cell.

Additional aspects and advantages will be apparent from the followingdetailed description of preferred embodiments, which proceeds withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show the chemical structures of heme (FIG. 1A),bilirubin IXα (FIG. 1B), and biliverdin IXα (FIG. 1C).

FIG. 2 shows the metabolic transformations of heme, biliverdin IXα andbilirubin IXα in animals.

FIG. 3 shows the roles of biliverdin IXα and HO-1 in the biosynthesis ofcyanobacterial phycocyanobilins.

FIG. 4 shows the cytoprotective role of biliverdin IXα and bilirubin IXαagainst ROS. Acronym definitions are: HO (heme oxygenase), BVR(biliverdin reductase) and ROS (reactive oxygen species). The barsindicate suppression of ROS.

FIG. 5 shows a gene map for pET101 plasmid expression vector with T7promoter used to express HemA and HO1 in E. coli.

FIG. 6 shows the DNA base sequence of cyanobacterium SynechocystisPCC6803 HO1 (SEQ ID NO: 48) (726 base pairs).

FIG. 7 shows the DNA base sequence of HemA of R. sphaeroides ALAsynthetase (SEQ ID NO: 49) (1224 base pairs).

FIG. 8 shows the chemical structure ofisopropyl-beta-D-thiogalactopyranoside (IPTG).

FIG. 9 shows a gene map of the plasmid expression vector HemA-HO1-pET101with HO1 and HemA genes inserted downstream of DNA sequences that permittranscriptional regulation via T7 lac operon dependent mechanisms andribosome binding sites (RBSs) for each gene to provide efficientinitiation of protein translation.

FIG. 10 shows a photograph of solid phase extraction Sepak C18 column (3ml) with green material produced by E. coli (HO-1). A methanol extractof green aggregated material from growing cultures was loaded in 40%methanol, 0.2M Na acetate, pH 5.2. The green material was then elutedoff the column with 100% methanol and recovered.

FIG. 11 shows absorbance spectra of a) methanol-extracted green materialproduced by E. coli (HO1), loaded onto a C18 solid phase extractionSepak C-18 column (3 ml) in 40% methanol, 0.2 M Na acetate, pH 5.2, andrecovered by elution with 100% methanol, and b) biliverdin IXα standard,commercially available from Frontier Scientific, Inc.

FIG. 12A shows a high-performance liquid chromatograms ofmethanol-extracted green material from cultures of E. coli (HO1).

FIG. 12B shows a high-performance liquid chromatograms of a biliverdinIXα standard, commercially available from Frontier Scientific, Inc. Thebacterially-derived green material (FIG. 12A) contains components withretention times that are identical to commercially available biliverdinIXα.

FIG. 13A shows a mass spectrum showing the mass of the methanolextracted and solid phase extraction recovered “green material” from E.coli (HO1).

FIG. 13B shows a mass spectrum showing the mass of a commercialbiliverdin IXα standard obtained from Frontier Scientific, Inc. Themethanol extracted and solid phase extraction recovered “green material”from E. coli (HO1) (FIG. 13A) has the same mass (m/e 583.68) as acommercial biliverdin IXα standard obtained from Frontier Scientific,Inc. (FIG. 13B).

FIG. 14 shows an overall strategy of increasing heme oxygenase activityand the biosynthesis of heme in a host cell to enhance Biliverdin IXalpha accumulation.

FIG. 15 shows dissolved oxygen (DO-1) and pH (pH-1) in growth medium ofEscherichia coli cells during biliverdin production.

FIG. 16A and FIG. 16B show an alignment of several HO family hemeoxygenases.

FIG. 17A shows an alignment of the HO-1 subfamily of heme oxygenases.FIG. 17B through FIG. 17C shows an alignment of the HMOX1 subfamily ofheme oxygenases. FIG. 17D shows an alignment of the ho subfamily of hemeoxygenases.

FIG. 18A, FIG. 18B, and FIG. 18C show an alignment of diverse ALAsynthases.

FIG. 19A, FIG. 19B, and FIG. 19C show an alignment of ALA synthases thatare homologs of SEQ ID NO: 2.

FIG. 20 shows the optimized HO-1 gene DNA sequence provided by DNA 2.0Inc. (SEQ ID NO: 50).

FIG. 21 shows the polypeptide sequence of HO-1 (SEQ ID NO: 1)

FIG. 22 shows a gene map for the vector HO1-pJ401.

FIG. 23 shows the polypeptide sequence of HemA (SEQ ID NO: 2).

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Biliverdin” means biliverdin IXα (CAS Registry Number: 114-25-0).

To “culture” or “culturing” means to provide nutrients to a cellsufficient to allow the cell to grow and reproduce. Methods of culturingcells are known in the art. In particular, method of culturing cells,including bacteria, are described in (Joe Sambrook, Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,2001 (ISBN: 0879695773); or in Frederick M. Ausubel, et al., eds.,Current Protocols in Molecular Biology, Wiley (ISSN: 1934-3639, lastupdated Jun. 28, 2010)), which are incorporated by reference.

“Recombinant” may be used to describe a polynucleotide or a polypeptide.A “recombinant polynucleotide” is a polynucleotide within a cell thatcomprises a sequence not naturally found inside that cell. For example,a recombinant polynucleotide could comprise a coding sequence (CDS) thatis not naturally found in the cell. Alternatively a recombinantpolynucleotide could comprise two sequences joined together, where onesequence is naturally found in the cell, and the other is not. Forexample a CDS naturally found in the cell could be operably linked to apromoter that is not naturally found in the cell. A “recombinantpolypeptide” is the polypeptide product of a recombinant polynucleotidethat comprises a CDS. For example, a recombinant heme oxygenase is thepolypeptide product of a recombinant polynucleotide which codes for theheme oxygenase.

A polynucleotide “codes for” a polypeptide when the polynucleotidecomprises a set of codons which, when transcribed and translated bycellular machinery, will produce a polypeptide whose amino acid sequencecorresponds to the codons of the polynucleotide according to a geneticcode.

“CDS” means coding sequence. A coding sequence is a polynucleotidesequence that codes for a polypeptide product.

A polynucleotide or polypeptide is “naturally found” in a cell when thatpolynucleotide or polypeptide is present in a healthy, uninfected,wild-type cell under one or more culture conditions. All otherpolynucleotides and polypeptides are not naturally found in a cell.

A “heme oxygenase” is an enzyme with the activity defined by EnzymeCommission (“E.C.”) number 1.14.99.3. Heme oxygenases include twofamilies of enzymes, the HO family, and the HemS family. The HO familyis a defined enzyme family comprising, for example, polypeptides (SEQ IDNOs:1 and 7-28). The identity of these SEQ IDs is shown in Table 1. TheHO family may be defined, for example, by shared sequence motifs asdescribed in the Hidden Markov Model Pf01126. An alignment of several HOfamily heme oxygenases is provided in FIG. 16. The HO family may also besubdivided into subfamilies, including the HMOX1 subfamily found in someanimals, the HO-1 subfamily found in some cyanobacteria and the hosubfamily found in some plants. An alignment of several subfamilymembers for several HO subfamilies is found in FIG. 17. The HemS familyof enzymes is also a defined enzyme family comprising Escherichia coliO157:H7 gene product ChuS and other proteins. The HemS family may bedefined, for example, by shared sequence motifs as described in theHidden Markov Model PF05171.

TABLE 1 Representative sample of HO family heme oxygenases SEQ IDSpecies Gene product 1 Synechocyctis PCC 6803 HO-1 7 Cyanotheca sp.cce2573 8 Synechococcus sp. A2508 9 Anabaena sp. all1897 10Corynebacterium Diphtheriae Hmuo 11 Synechocystis sp. 1WOW_A 12Oryctolagus cuniculus heme oxygenase 2 13 Pseudomonas aeruginosa gi50513550 14 Takifugu rubripes HMOX 15 Drosophila melanogaster Q9VGJ9 16Bradyrhizobium sp gi 75412672 17 Pseudomonas aeruginosa gi 81540044 18Streptomyces coelicolor gi 81550417 19 Bos taurus HMOX1 20 Homo sapiensHMOX1 21 Mus musculus Hmox1 22 Rattus norvegicus Hmox1 23 Arabidopsisthaliana ho4 24 Arabidopsis thaliana ho3 25 Gallus gallus HMOX1 26 Daniorerio hmox1 27 Pan troglodytes HMOX1 28 Canis lupus familiaris HMOX1

A “heme oxygenase analog” means a heme oxygenase enzyme bearing one ormore additions deletions or substitutions of residues compared to theoriginal heme oxygenase. Useful heme oxygenase analogs include analogswhich retain the heme oxygenase activity defined by E.C. 1.14.99.3. Byexamining and aligning known heme oxygenase sequences from a given hemeoxygenase family, a skilled person can determine which heme oxygenaseresidues are conserved across species. Using this alignment, the skilledperson could generate a consensus sequence, using, for example, theClustal algorithm. Since conserved residues are generally those whichare required for function (Boffelli D, Nobrega M A, Rubin E M.Comparative genomics at the vertebrate extremes. Nat Rev Genet. 2004;5:456-465), non-naturally occurring proteins that conform to thisconsensus sequence would define heme oxygenase analogs that likelyretain the heme oxygenase activity defined by E.C. 1.14.99.3. Forexample, from the alignments in FIG. 16 or 17, a consensus sequence forthe HO family could be constructed. Further, additional alignments maybe generated using, for example, the HomoloGene and Conserved Domain(CDD) algorithms of the National Center for Biotechnology Information(NCBI), U.S. National Library of Medicine, Bethesda Md. USA.

Alternatively, generating heme oxygenase analogs which retain hemeoxygenase activity could also be accomplished by using existingbioinformatic resources. Proteins and protein domains are oftendescribed by a Hidden Markov Model (HMM). An HMM of a polypeptide is nota sequence alignment, but it does convey actual structural informationabout the protein. Most HMMs are based on the probability of anyparticular residue occurring next to a second residue in the linearsequence of the polypeptide. Using HMMs to describe proteins isdiscussed in Krogh A, Brown M, Mian I S, Sjölander K, Haussler D, HiddenMarkov models in computational biology. Applications to proteinmodeling. J Mol Biol. 1994; 235; 1501-31, which is hereby incorporatedby reference.

The European Bioinformatics Institute maintains the Interpro database,which compiles HMM information from various databases, including somedescribed below. Interpro has two different entries which describe hemeoxygenases. The first is IPR002051 Haem oxygenase (defining the HOfamily) and IPR007845 Haemin-degrading HemS (defining the HemS family).

The Wellcome Trust Sanger Institute also maintains the Pfam database,which describes the heme oxygenase proteins in terms of HMMs. The PfamHMMs that define the heme oxygenase proteins are PF01126 (HO family) andPF05171 (HemS family). Included in the database for each HMM entry is afeature which allows the user to visualize the structural information inthe HMM.

Although the HMMs do not provide typical sequence information regardingheme oxygenase proteins, they do provide a description of the probablestructure of a heme oxygenase. Thus, analogs of heme oxygenase thatconform to the HMM would be more likely to retain heme oxygenaseactivity. To easily generate sequences of heme oxygenase analogs morelikely to have heme oxygenase activity, a skilled person could generateheme oxygenase analog sequences using a computer to introducesubstitutions, deletions or additions to a heme oxygenase sequence. Therelative probabilities embodied in the heme oxygenase HMMs could guide askilled person regarding which residues, when mutated, are more likelyto lead to a loss of function. The skilled person could then compare theanalog sequences to the HMMs in the databases listed above. Thoseanalogs which met the threshold of being tagged as bearing a hemeoxygenase domain would likely have the property of heme oxygenaseactivity. The HMMs discussed above which describe heme oxygenases andheme oxygenase analogs are hereby incorporated by reference.

“HO1” or “HO-1” is the polypeptide represented by SEQ ID NO: 1.

A “heme biosynthetic enzyme” is an enzyme involved in the anabolicmetabolism of heme. Heme biosynthetic enzymes include Amino levulinicacid dehydratase, Porphobilinogen deaminase, Uroporphyrinogen IIIsynthase, Uroporphyrinogen III decarboxylase, Coprophorinogen IIIoxidase, Protopophyrinogen IX oxidase, and Ferrochetalase. These enzymesare well characterized and their role in heme biosynthesis isunderstood. A very important step in the production of Heme is theproduction of amino levulinic acid (ALA). Two anabolic pathways existfor the production of ALA, the C-4 and C-5 pathways. The enzymes of theC-4 and C-5 pathway are heme biosynthetic enzymes. An example of a C-5pathway enzyme is glutamyl-tRNA reductase. An example of a C-4 pathwayenzyme is ALA synthase.

“An “ALA synthase” or “ALA synthetase” is an enzyme with the activitydefined by E.C. 2.3.1.37. ALA synthase enzymes are not subject tofeedback inhibition from heme. ALA synthases are a defined class ofenzymes including, for example, polypeptides (SEQ ID NOs: 2 and 29-47).The identity of these SEQ IDs is shown in Table 2. The ALA synthases maybe defined, for example, by shared sequence motifs as described in theHidden Markov Model TIGR01821. An alignment of diverse ALA synthases isprovided in FIG. 18. An alignment of conserved homologs of SEQ ID NO: 2is provided in FIG. 19.

TABLE 2 Representative sample of ALA synthetases SEQ ID Species Geneproduct 2 Rhodobacter sphaeroides 2.4.1 HemA 29 Hyphomonas neptuniumATCC 15444 gi 114797766 30 Orientia tsutsugamushi str.Boryong gi148284187 31 Azorhizobium caulinodans ORS 571 gi 158421958 32Caulobacter crescentus CB15GI:16125604 gi 16125604 33 Brucella canisATCC 23365 gi 161618302 34 Bordetella petrii DSM 12804 gi 163855632 35Caulobacter sp. K31 gi 167647011 36 Streptomyces griseus subsp.griseusgi 182439088 37 Orientia tsutsugamushi str.Ikeda gi 189183979 38Phenylobacterium zucineum HLK1 gi 197105140 39 Phenylobacterium zucineumHLK1 gi 197106256 40 Caulobacter crescentus NA1000 gi 221234354 41Candidatus Liberibacter asiaticus str. gi 254780604 42 Neorickettsiaristicii str. gi 254797163 43 Brucella microti CCM 4915 gi 256368778 44Chromobacterium violaceum ATCC 12472 gi 34496258 45 Brucella abortus bv.1 str. gi 62289313 46 Staphylococcus aureus RF122 gi 82751601 47Neorickettsia sennetsu str.Miyayama gi 88608338

An “ALA synthase analog” means an ALA synthase enzyme bearing one ormore additions deletions or substitutions of residues compared to theoriginal ALA synthase. Useful ALA synthase analogs include analogs whichretain the activity defined by E.C. 2.3.1.37. By examining and aligningknown ALA synthase sequences, a skilled person can determine which ALAsynthase residues are conserved across species. Using this alignment,the skilled person could generate a consensus sequence, using, forexample, the Clustal algorithm. Since conserved residues are generallythose which are required for function (Boffelli D, Nobrega M A, Rubin EM. Comparative genomics at the vertebrate extremes. Nat Rev Genet. 2004;5:456-465), non-naturally occurring proteins that conform to thisconsensus sequence would define ALA synthase analogs that likely retainthe ALA synthase activity defined by E.C. 2.3.1.37. For example, fromthe alignment in FIG. 18 or 19, a consensus sequence for several ALAsynthases may be constructed. Further, additional alignments may begenerated using, for example, the HomoloGene and Conserved Domain (CDD)algorithms of the National Center for Biotechnology Information (NCBI),U.S. National Library of Medicine, Bethesda Md. USA.

Alternatively, generating ALA synthase analogs which retain ALA synthaseactivity could also be accomplished by using HMM as described above.

Interpro has one entry which describe ALA synthases: IPR010961Tetrapyrrole biosynthesis, 5-aminolevulinic acid synthase. The J. CraigVenter Institute maintains the TIGR database. The TIGR HMM profile thatdescribes the probable structure of ALA synthases is TIGR01821.

Although these HMMs do not provide typical sequence informationregarding ALA synthase proteins, they do provide a description of theprobable structure of a ALA synthase. Thus, analogs of ALA synthase thatconform to the HMM would be more likely to retain ALA synthase activity.To easily generate sequences of ALA synthase analogs more likely to haveALA synthase activity, a skilled person could generate ALA synthaseanalog sequences using a computer to introduce substitutions, deletionsor additions to a ALA synthase sequence. The relative probabilitiesembodied in the ALA synthase HMMs could guide a skilled person regardingwhich residues, when mutated, are more likely to lead to a loss offunction. The skilled person could then compare the analog sequences tothe HMMs in the databases listed above. Those analogs which met thethreshold of being tagged as bearing a ALA synthase domain would likelyhave the property of ALA synthase activity. The HMMs discussed abovewhich describe ALA synthases and ALA synthase analogs are herebyincorporated by reference.

“HemA” is the polypeptide represented by SEQ ID NO: 2.

“Growth media” or “growth medium” is a composition comprising one ormore nutrients used to culture a cell. Growth medium includes any foamwhich accumulates in the medium.

A “vector” is a polynucleotide which can be used to introduce a desiredsequence into a cell. The vector polynucleotide typically includesadditional sequences, including sequences that direct propagation of thevector in the cell or insertion of part of the vector into the cell'sgenome, and a gene which allows an individual to screen for the presenceof the vector. Common examples of vectors include plasmids, artificialchromosomes, viruses, and linear polynucleotide fragments which aredesigned to insert into a cell's genome. Vectors are well known tools tothe skilled artisan, and an artisan can easily find appropriate vectorsfor a particular organism in the literature or in biobanks such as ATCC.

A “promoter” is a polynucleotide sequence that, when operably linked toa CDS, is sufficient, under one or more conditions, to cause an RNApolymerase to begin transcribing an mRNA from a polynucleotide.

A promoter is “operably linked” to a polynucleotide sequence when thepromoter is linked to the polynucleotide sequence in such a way that aRNA polymerase will transcribe at least a portion of the polynucleotidesequence. Typically, linking a promoter to the 5′ end of apolynucleotide will result in the promoter being operably linked to thepolynucleotide.

In the context of a first polynucleotide and a second polynucleotide,the first and second polynucleotides are “linked” when they are joinedby a linker or a phosphodiester bond.

A “linker” is a polynucleotide of one of more bases which is used tolink two or more polynucleotides.

“Foam” is a suspension of a gas in another substance. When foamaccumulates in growth media, a gas which is in contact with the growthmedia become suspended in the media. For example foam often forms invessels containing growth media when they are agitated.

Biliverdin is from a “non-animal source” when the heme oxidase catalyzedstep of biliverdin production is not performed in a cell from anorganism in the Eukaryotic kingdom animalia, and where the hemesubstrate of the heme oxidase was not produced in a cell from anorganism in the Eukaryotic kingdom animalia.

“Collecting” a substance means removing a measurable quantity of thesubstance from the vessel in which it was produced. For example, if foamwas produced in a vessel while a cell was cultured, removing the foamfrom the vessel is collecting the foam. Likewise, in the context ofproducing biliverdin, removing biliverdin from the vessel in which itwas produced is collecting the biliverdin. “Collecting” does notnecessitate purification of the biliverdin from the organism in whichthe biliverdin was synthesized.

In the context of generating a cell for producing biliverdin, “a parentcell” is a cell into which a recombinant heme oxygenase and/or arecombinant heme biosynthetic enzyme is introduced.

To “introduce” or “introducing” a polynucleotide into a cell means usingphysical or genetic techniques to cause the polynucleotide to enterwithin a membrane which surrounds the cell. These includetransformation, conjugation between bacterial cells, and recombinationduring sexual reproduction.

To “transform” or “transforming” means to introduce a polynucleotideinto a cell by chemical, electrical or other physical means. Methods oftransforming cells include chemical destabilization of the cell membraneto allow the polynucleotide to enter, electroporation, microinjection,or firing particles coated with the polynucleotide into the cell.

A composition found inside a cell is “excreted” when the compositionmoves outside of the membrane surrounding the cell. A composition may beexcreted passively, meaning that the composition diffuses across themembrane, or a compound may be excreted actively, meaning that cellularfactors aid the composition in moving outside the membrane surroundingthe cell.

A first composition is “located with” a second composition when ameasurable quantity of the second composition is associated with thefirst composition such that collecting the first composition will alsocollect some of the associated second composition.

A polynucleotide that contains a CDS is “optimized for expression in acell” where one or more nucleotide bases of the polynucleotide aremodified according to an algorithm such that when the polynucleotide islinked to a promoter and introduced into a cell, more of the proteinproduct of the CDS is produced than would be produced by the unmodifiedpolynucleotide linked to the same promoter.

Various methods of modifying a polynucleotide so that it is optimizedfor expression in a cell are known. For example, the polynucleotidesequence may be modified according to the Codon Adaptation Index method(Sharp, P. M., and W. H. Li, (1987). The codon adaptation index ameasure of directional synonymous codon usage bias, and its potentialapplications. Nucleic Acids Research 15: 1281-1295, which is herebyincorporated by reference). Also, the polynucleotide sequence may bemodified according to the Frequency of Optimal Codons method (Ikemura,T., (1981). Correlation between the abundance of Escherichia colitransfer RNAs and the occurrence of the respective codons in its proteingenes: a proposal for a synonymous codon choice that is optimal for theE. coli system. Journal of Molecular Biology 151: 389-409, which isincorporated by reference).

Alternatively, a polynucleotide may be optimized for expression in acell where the nucleotide bases are modified according to theproprietary methods of DNA 2.0 Inc., Menlo Park, Calif.

Many of the embodiments described below incorporate methods forculturing cells, transforming cells, or performing other geneticmanipulations on cells. Many methods for performing these steps areknown in the art. In particular, many laboratory methods are describedin Joe Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, 2001 (ISBN: 0879695773); or inFrederick M. Ausubel, et al., eds., Current Protocols in MolecularBiology, Wiley (ISSN: 1934-3639, last updated Jun. 28, 2010, which areincorporated by reference.

Methods of Producing Biliverdin

One aspect of the invention includes methods of producing biliverdin ina microorganism. Another aspect of the invention includes methods forproducing biliverdin from a non-animal source. In general, both methodsof producing biliverdin in a microorganism and methods of producingbiliverdin from a non-animal source include culturing a cell comprisinga recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.The embodiments described below represent embodiments of both methods ofproducing biliverdin in a microorganism and methods for producingbiliverdin from a non-animal source.

In one embodiment, the cultured cell comprises a vector comprising apolynucleotide which codes for a recombinant heme oxygenase. In anotherembodiment, the cell comprises a vector comprising a polynucleotidewhich codes for a recombinant heme biosynthetic enzyme. The vector maybe a plasmid, a construct designed to integrate into the genome of thecell, an artificial chromosome, or any other vector known in the artwhich is appropriate for use in the cell. For example, ATCC maintains acollection of vectors for use in a variety of organisms.

In another embodiment, a promoter may drive expression of apolynucleotide which codes for a recombinant heme oxygenase. In anotherembodiment, a promoter may drive expression of a polynucleotide whichcodes for a recombinant heme oxygenase. In any embodiment where apromoter is used, the promoter may be a regulatable promoter.Furthermore, regulatable promoters may be used to control the expressionof recombinant polypeptides in a temporal or other fashion. Someregulatable promoters are inducible promoters. For example, the T7promoter drives very low basal levels of expression when cells are grownin the absence of IPTG. However, when IPTG is added to the culturemedia, the promoter is activated and higher expression is induced. Otherregulatable promoters may be repressible promoters. For example, thetetR promoter has very low expression when cells are grown in thepresence of tetracycline, but expression increases when tetracycline isremoved from the growth medium. Many promoters which are appropriate foruse in a variety of cells are known in the art. The Registry of StandardBiological Parts, maintained by Massachusetts Institute of Technologydiscloses many promoters which will be appropriate for use in differenttypes of cells to achieve a desired pattern of expression. The Registryof Standard Biological Parts is hereby incorporated by reference.

In another embodiment, the heme oxygenase enzyme is a HO family hemeoxygenase. In another embodiment, the heme oxygenase is a HemS familyheme oxygenase. In a particular embodiment, HO1 (SEQ ID NO: 1) is usedas the heme oxygenase. In another embodiment, the heme oxygenase enzymemay be an HO family analog.

In another embodiment, the heme biosynthetic enzyme used is ALAdehydratase. In another embodiment, the heme biosynthetic enzyme used isPorphobilinogen deaminase. In another embodiment, the heme biosyntheticenzyme used is Uroporphyrinogen III synthase. In another embodiment, theheme biosynthetic enzyme used is Uroporphyrinogen III decarboxylase. Inanother embodiment, the heme biosynthetic enzyme used is CoprophorinogenIII oxidase. In another embodiment, the heme biosynthetic enzyme used isProtopophyrinogen IX oxidase. In another embodiment, the hemebiosynthetic enzyme used is Ferrochetalase.

In certain embodiments, the heme biosynthetic enzyme used is an ALAsynthase. In a particular embodiment, the heme biosynthetic enzyme ishemA (SEQ ID NO: 2). In certain other embodiments, the heme biosyntheticenzyme is an ALA synthase analog.

In a particular embodiment, the cell comprises HemA-HO1-pET101 (FIG. 9).

In another embodiment, lactose is provided to the cell which is beingcultured. Generally, lactose may be provided to the cell as a componentof the growth medium. Lactose may be provided in a initialconcentration, and the actual concentration of lactose in the media willdecrease as the cells consume it. In one embodiment, the initialconcentration of lactose is from about 2% (w/v) to about 10% (w/v). Inanother embodiment, the lactose may be provided in an initialconcentration of about 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or about 10%. Ina particular embodiment the initial concentration of lactose is 2.5%.

In another embodiment, one or more trace metals is provided to the cell.The trace metal(s) may be provided in the form of a trace metalsolution. In one embodiment, one or more of NaCl, ZnSO₄, MnCl₂, FeCl₃,CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine or CaCl₂ are added to theculture. In another embodiment, NaCl, ZnSO₄, MnCl₂, FeCl₃, CuSO₄, H₃BO₃,NaMoO₄, H₂SO₄, MgSO₄, thiamine and CaCl₂ are added to the culture.

In another embodiment, the concentration of dissolved oxygen in thegrowth medium of the cell is measured. Any oxygen measuring device knownin the art can be used. For example an oxygen measuring probe may beused. In one embodiment, InPro® 6800 Series O₂ Sensors (METTLER TOLEDO)may be used in monitoring the concentration of dissolved oxygen In oneembodiment, a plurality of measurements of the dissolved oxygenconcentration are made. In another embodiment, the concentration ofdissolved oxygen has an initial value, then the dissolved oxygenconcentration decreases to a first intermediate value, then thedissolved oxygen concentration increases to a second intermediate valuegreater than the first intermediate value, then the dissolved oxygenconcentration decreases to a third intermediate value less than thefirst intermediate value.

In another embodiment, the biliverdin is excreted from the cell into thegrowth medium. In another embodiment, foam accumulates in the growthmedium, and a portion of the biliverdin is located with the foam. In arelated embodiment, the foam is collected. In another embodiment, thebiliverdin is collected.

For any embodiment of a method of producing biliverdin discussed above,the cell may comprise a polynucleotide that is optimized for expressionin a cell

In certain embodiments, the cell comprises a sequence that codes for aheme oxygenase that is optimized for expression in a cell. In certainembodiments, the cell comprises a sequence that codes for an HO familyheme oxygenase that is optimized for expression in a cell. In oneembodiment, the cell comprises a sequence that codes for HO-1 that isoptimized for expression in a cell. In one embodiment, the cellcomprises a sequence that codes for HO-1 that is optimized forexpression in a bacterial cell. In one embodiment, the cell comprises asequence that codes for HO-1 that is optimized for expression in anEscherichia coli cell. In another embodiment, the cell comprises apolynucleotide comprising SEQ ID NO: 50, wherein the cell is a bacterialcell. In another embodiment, the cell comprises a polynucleotidecomprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is abacterial cell. In another embodiment, the cell comprises apolynucleotide comprising SEQ ID NO: 50, wherein the cell is anEscherichia coli cell. In another embodiment, the cell comprises apolynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein thecell is an Escherichia coli cell.

In certain embodiments, the cell comprises a sequence that codes for aheme biosynthetic enzyme that is optimized for expression in a cell. Incertain embodiments the cell comprises a sequence that codes for an ALAsynthase that is optimized for expression in a cell. In one embodiment,the cell comprises a sequence that codes for HemA that is optimized forexpression in a cell. In a related embodiment, the cell comprises asequence that codes for HemA that is optimized for expression in abacterial cell. In a related embodiment, the cell comprises a sequencethat codes for HemA that is optimized for expression in an Escherichiacoli cell.

Cells for Producing Biliverdin

Another aspect of the invention includes cells for producing biliverdin.In general, cells for producing biliverdin comprise a recombinant hemeoxygenase and a recombinant heme biosynthetic enzyme.

In one embodiment, the cell comprises a vector comprising apolynucleotide which codes for a recombinant heme oxygenase. In anotherembodiment, the cell comprises a vector comprising a polynucleotidewhich codes for a recombinant heme biosynthetic enzyme. The vector maybe a plasmid, a construct designed to integrate into the genome of thecell, an artificial chromosome, or any other vector known in the artwhich is appropriate for use in the cell. For example, ATCC maintains acollection of vectors for use in a variety of organisms.

In another embodiment, the cell comprises a promoter that may driveexpression of a polynucleotide which codes for a recombinant hemeoxygenase. In another embodiment, the cell comprises a promoter that maydrive expression of a polynucleotide which codes for a recombinant hemeoxygenase. In any embodiment where a promoter is used, the promoter maybe a regulatable promoter. Furthermore, regulatable promoters may beused to control the expression of recombinant polypeptides in a temporalor other fashion. Some regulatable promoters are inducible promoters.For example, the T7 promoter drives very low basal levels of expressionwhen cells are grown in the absence of IPTG (FIG. 8). However, when IPTGis added to the culture media, the promoter is activated and higherexpression is induced. Other regulatable promoters may be repressiblepromoters. For example, the tetR promoter has very low expression whencells are grown in the presence of tetracycline, but expressionincreases when tetracycline is removed from the growth medium. Manypromoters which are appropriate for use in a variety of cells are knownin the art. The Registry of Standard Biological Parts, maintained byMassachusetts Institute of Technology discloses many promoters whichwill be appropriate for use in different types of cells to achieve adesired pattern of expression. The Registry of Standard Biological Partsis hereby incorporated by reference.

In another embodiment, the heme oxygenase enzyme is a HO family hemeoxygenase. In another embodiment, the heme oxygenase is a HemS familyheme oxygenase. In a particular embodiment, HO1 (SEQ ID NO: 1) is usedas the heme oxygenase. In another embodiment, the heme oxygenase enzymemay be an HO family analog.

In another embodiment, the heme biosynthetic enzyme used is ALAdehydratase. In another embodiment, the heme biosynthetic enzyme used isPorphobilinogen deaminase. In another embodiment, the heme biosyntheticenzyme used is Uroporphyrinogen III synthase. In another embodiment, theheme biosynthetic enzyme used is Uroporphyrinogen III decarboxylase. Inanother embodiment, the heme biosynthetic enzyme used is CoprophorinogenIII oxidase. In another embodiment, the heme biosynthetic enzyme used isProtopophyrinogen IX oxidase. In another embodiment, the hemebiosynthetic enzyme used is Ferrochetalase.

In certain embodiments, the heme biosynthetic enzyme used is an ALAsynthase. In a particular embodiment, the heme biosynthetic enzyme ishemA (SEQ ID NO: 2). In certain other embodiments, the heme biosyntheticenzyme is an ALA synthase analog.

In a particular embodiment, the cell comprises HemA-HO1-pET101 (FIG. 9).

For any embodiment that provides a cell for producing biliverdindiscussed above, the cell may comprise a polynucleotide that isoptimized for expression in a cell.

In certain embodiments, the cell comprises a sequence that codes for aheme oxygenase that is optimized for expression in a cell. In certainembodiments, the cell comprises a sequence that codes for an HO familyheme oxygenase that is optimized for expression in a cell. In oneembodiment, the cell comprises a sequence that codes for HO-1 that isoptimized for expression in a cell. In one embodiment, the cellcomprises a sequence that codes for HO-1 that is optimized forexpression in a bacterial cell. In one embodiment, the cell comprises asequence that codes for HO-1 that is optimized for expression in anEscherichia coli cell. In another embodiment, the cell comprises apolynucleotide comprising SEQ ID NO: 50, wherein the cell is a bacterialcell. In another embodiment, the cell comprises a polynucleotidecomprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is abacterial cell. In another embodiment, the cell comprises apolynucleotide comprising SEQ ID NO: 50, wherein the cell is anEscherichia coli cell. In another embodiment, the cell comprises apolynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein thecell is an Escherichia coli cell.

In certain embodiments, the cell comprises a sequence that codes for aheme biosynthetic enzyme that is optimized for expression in a cell. Incertain embodiments the cell comprises a sequence that codes for an ALAsynthase that is optimized for expression in a cell. In one embodiment,the cell comprises a sequence that codes for HemA that is optimized forexpression in a cell. In a related embodiment, the cell comprises asequence that codes for HemA that is optimized for expression in abacterial cell. In a related embodiment, the cell comprises a sequencethat codes for HemA that is optimized for expression in an Escherichiacoli cell.

Methods of Producing Cells

Another aspect of the invention includes methods of producing cells forproducing biliverdin. In general, these methods comprise introducinginto a cell a recombinant polypeptide. In one embodiment, a recombinantheme oxygenase is introduced into the cell. In another embodiment, arecombinant heme biosynthetic enzyme is introduced into the cell.

In one embodiment, the method comprises introducing into the cell apolynucleotide comprising a sequence which codes for a recombinant hemeoxygenase. In another embodiment, the method comprises introducing intothe cell a polynucleotide comprising a sequence which codes for arecombinant heme biosynthetic enzyme. In another embodiment, apolynucleotide is introduced into the cell, the polynucleotidecomprising a first sequence which codes for a recombinant heme oxygenaseand a second sequence which codes for a recombinant heme biosyntheticenzyme. In a particular embodiment, the HemA-HO1-pET101 (FIG. 9) isintroduced into the cell.

In another embodiment, a promoter is introduced into the cell in such away that it recombines with a sequence which codes for a heme oxygenaseto produce a linked polynucleotide which codes for a recombinant hemeoxygenase, where the parent cell comprises a recombinant hemebiosynthetic enzyme. In another embodiment, a promoter is introducedinto the cell in such a way that it recombines with a sequence whichcodes for a heme biosynthetic enzyme to produce a linked polynucleotidewhich codes for a recombinant heme biosynthetic enzyme, where the parentcell comprises a recombinant heme oxygenase. Methods of introducingpolynucleotides for directed recombination with polynucleotides in acell are known in the art. For example, methods for directedrecombination are discussed in A J Link et al., Methods for generatingprecise deletions and insertions in the genome of wild-type Escherichiacoli: application to open reading frame characterization, J. Bacteriol,(179) 6228-6237 (1997), which is incorporated by reference.

In another embodiment, any step comprising introducing a polynucleotideinto a cell comprises transforming the cell.

In another embodiment, the cell is a microorganism. In anotherembodiment, the cell is a prokaryotic cell. In another embodiment, thecell is a bacterial cell. In another embodiment, the cell is anEscherichia coli cell.

For any embodiment that provides a method of producing a cell forproducing biliverdin discussed above, the polynucleotides may beoptimized for expression in a cell.

In certain embodiments, a sequence that codes for a heme oxygenase thatis optimized for expression in a cell is introduced into a cell. Incertain embodiments, a sequence that codes for an HO family hemeoxygenase that is optimized for expression in a cell is introduced intoa cell. In one embodiment, a sequence that codes for HO-1 that isoptimized for expression in a cell is introduced into a cell. In anotherembodiment, a polynucleotide comprising SEQ ID NO: 50 is introduced intoa bacterial cell. In another embodiment, a polynucleotide comprisingbases 15 to 776 of SEQ ID NO: 50 is introduced into a bacterial cell. Inanother embodiment, a polynucleotide comprising SEQ ID NO: 50 isintroduced into an Escherichia coli cell. In another embodiment, apolynucleotide comprising bases 15 to 776 of SEQ ID NO: 50 is introducedinto an Escherichia coli cell.

In certain embodiments, a sequence that codes for a heme biosyntheticenzyme that is optimized for expression in a cell is introduced into acell. In certain embodiments a sequence that codes for an ALA synthasethat is optimized for expression in a cell is introduced into a cell. Inone embodiment, a sequence that codes for HemA that is optimized forexpression in a cell is introduced into a cell. In a related embodiment,a sequence that codes for HemA that is optimized for expression in acell is introduced into a bacterial cell. In a related embodiment, asequence that codes for HemA that is optimized for expression in a cellis introduced into an Escherichia coli cell.

The following examples are intended to further illustrate exemplaryembodiments and are not intended to limit the scope of the disclosure.

EXAMPLES Example 1 Construction of Plasmid Expression Vectors and GeneExpression

HO-1 pET101: HO (HO-1) gene of Synechocystis PCC6803 was amplified bythe polymerase chain reaction (PCR) using the following primers:

(SEQ ID NO: 3) (HO1 forward primer)- CACCATGAGTGTCAACTTAGCTTC(SEQ ID NO: 4) (HO1 reverse primer)- CTAGCCTTCGGAGGTGGCGA

The PCR product was blunt ended using thermostable proofreadingpolymerase, gel purified, ligated into pET101 vector by directionalTOPO® Cloning Reaction and transformed into chemically competent E. coliTOP10 (Invitrogen) cells according to the manufacturer's instructions.Five white colonies were selected on Xgal agar plates, plasmids isolatedand subjected to gel electrophoresis to confirm cloning of HO-1 into thevector. DNA sequencing showed that the cloned DNA had an identicalsequence to Synechococcus PCC6803 HO-1 (SEQ ID NO: 48) (FIG. 6). Thevector with the HO-1 gene was transformed into E. coli BL21 (Invitrogen)and cells from a single white colony were propagated in Luria-Bertani(LB) broth medium (25 g per L, Fisher Scientific) plus 100 μg per mLampicillin., Its DNA plasmids were isolated and the HO-1gene sequence inthe plasmid was confirmed by DNA sequencing again. The plasmid was usedas the clone that harbored expression vector HO-1 pET101.

HemA-HO-1 pET101: HemA which encodes ALA synthase from R. sphaeroideswas amplified by the polymerase chain reaction (PCR) using the followingprimers:

(SEQ ID NO: 5) (Hem A forward primer)-ACAACGTTGAAGGAGCCCTTCTCCATGGACTACAATCTGGCACT (SEQ ID NO: 6)(Hem A reverse primer)- ATGACCGGTACGTCAGGCAACGACCTCGGCGC

The HemA gene was cut by restriction enzymes (AdII and AgeI) and ligatedto HO1-pET101 vector which was digested by restriction enzymes (BstBIand AgeI). The construct was transformed into competent E. coli BL21(DE3) (Invitrogen) cells according to the manufacturer's instructions.Five white colony isolates were selected and were propagated inLuria-Bertani (LB) broth medium (25 g per L, Fisher Scientific) plus 100μg per mL ampicillin. Their plasmid DNAs were extracted and theoccurrence of HemA was confirmed by DNA sequencing (FIG. 7—SEQ ID NO:49). After DNA sequencing analysis of the plasmid DNAs, an isolateyielding plasmid DNA with the expected size was selected, designated E.coli strain HemA-HO-1 and was used as the clone that harbors expressionvector HemA-HO-1 pET101.

Example 2 Bacterial Growth, Protein Expression, and Production ofBiliverdin

E. coli strains HO-1 and HemA-HO-1 were maintained on LB agar mediumwith 100 μg per mL ampicillin. For analysis of protein expression, cellswere grown in LB broth medium supplemented with 1% glucose and 100 μgper mL ampicillin with rotary shaking 225 rpm at 37° C. overnight. IPTGwas added (1 mM final concentration) when the culture achieved anabsorbance between 0.3 and 0.5 at 600 nm. Exponentially grown cells wereharvested and lysed and cell extracts were recovered as supernatantfractions after centrifugation at 8,000×g for a minimum of 5 min. Thecell extracts were subjected to sodium dodecylsulfate polyacrylamide gelelectrophoresis and the gel was stained with Coomasie Brilliant Blue.The stained gel showed the induction of the HO-1 protein (23 kdaltons)by IPTG in both E. coli strains which confirmed the expression of HO-1gene.

For biliverdin production, the HemA and HO-1 containing E. coli strainsare grown in 100 to 200-ml of LB broth medium plus 100 μg per mLampicillin in 500-mL capacity Erlenmeyer flasks in a rotary water bathshaker (200 rpm) at 37° C. to a cell density showing an absorbance of 4to 5 measured at 600 nm using a 1 cm path length cuvette and LB brothmedium as blank. The culture is then added to 1 or 1.5 L of LB brothmedium or biliverdin-Minimal Medium (biliverdin 2) (per L, KH₂PO₄, 3.5g; K₂HPO₄, 5.0 g; (NH₄)₂SO₄, 5.0 g; yeast extract, 5.0 g; trace metalssolution (per L, NaCl, 5 g; ZnSO₄-7H₂O, 1 g; MnCl₂-4H₂O, 4 g;FeCl₃-6H₂O, 4.75 g; CuSO₄-5H₂O, 0.4 g; H₃BO₃, 0.575 g; NaMoO₄-2H₂O, 0.5g; and 6N H₂SO₄, ˜12.5 ml), 1 mL; MgSO₄-7H₂O (25%), 4 mL; thiamine (220mg per mL), 10 mL, CaCl₂—H₂O (15 g per L), 10 mL with trace metalssolution, MgSO₄-7H₂O, thiamine, and CaCl₂—H₂O filter sterilizedseparately and added to the other ingredients after autoclaving)containing 100 mg per L ampicillin and 2.5% α-lactose both filteredsterilized and added separately after autoclaving. The final pH for bothgrowth media is 7.0. The E. coli inocula cultures are added to give aninitial absorbance of ˜0.03 measured at 600 nm using a 1 cm path lengthcuvette. The cultures are grown in a VirTis Omniculture bioreactorsystem with 2.0 L vessel (VirTis, Gardner, N.Y.). Airflow is at 3.5liters per min at 30° C. Agitation is set at 250 rpm until an absorbanceof 0.4 to 0.5 measured at 600 nm (1 cm path length) is attained, andthen agitation is increased to 450 rpm. Under these conditions,blue-green pigmented material becomes visible 6 to 10 hours at or nearthe top of foam formed above the surface of the culture. At this point,dissolved oxygen levels are less than 5% saturation. The materialaccumulates as a blue-green film and as blue-green aggregates thatadhere to the glass walls of the vessel and the stainless steel headplate. In addition, polyethylene tubing (Tygon, ¼ inch I.D., 0.5 to 1 mlength) connected to an outlet port of the vessel head plate and withthe other end opened into a receiving flask is used to collectfoam-trapped blue-green material during culture growth. Growth isterminated after approximately 20 hours or when production of blue-greenmaterial ceases.

Example 3 Collecting the Biliverdin

After the growth of a batch culture is completed, the blue-green filmand aggregated materials are physically removed from the vessel surfacesusing a spatula and suspended in methanol. The methanol suspension iscombined with the blue green material collected in the receiving flaskand an equivalent volume of methanol is added. A volume of 1N HCl equalto the total volume of the methanol suspension is added dropwise to thesuspension with stirring. A 1/10 volume of water is added, and themixture is vortexed and extracted into chloroform. The green chloroformlayer is recovered, dried, and the resulting blue-green material isstored in the dark at −20° C. Absorbance spectra of this materialresemble the spectrum obtained for authentic biliverdin (FrontierScientific, Inc.) suggesting that the blue-green material containsbiliverdin IXα. For purification, the dried material is dissolved in 40%methanol, 0.2 M Na acetate, pH 5.2 and loaded onto a Sepak C18 column.The column is eluted successively with 40% methanol, 0.2 M Na acetate,pH 5.2, water, and 100% methanol. The blue-green material elutes with100% methanol (FIG. 10) and is collected. It is acidified with HCl (0.25N final concentration), extracted into chloroform and stored in the darkat −20° C.

Example 4 Analytical Determination of Biliverdin

Absorbance spectra between wavelengths 300 and 800 nm were obtainedusing an Applied Biosystem spectrophotometer. The presence of biliverdinin the blue-green material is evident by comparison to the absorbancespectrum of a known standard of biliverdin (from Frontier Scientific,Inc.) derived from bilirubin IXα (FIG. 11) with characteristic spectralpeaks at 385 nm and 650 nm-660 nm. The same absorbance spectrum wasobtained with blue-green material produced by E. coli strains TOP10 orBL21 transformed with and expressing HO1-pET101 or HemA-HO-1 pET101,respectively. Also, the same absorbance spectrum was obtained withblue-green material from cultures of E. coli strains grown in either LBbroth medium or biliverdin 2 broth medium. High performance liquidchromatography (HPLC) is also an analytical tool to help determine thechemical identity of the purified blue-green material as biliverdin.Identical or very similar HPLC peaks (major peaks with Rf values of13.19 and 13.17 in FIG. 12) monitored at 385 nm are obtained with theblue-green material produced by E. coli strains TOP10 or BL21transformed with and expressing HO1-pET101 or HemA-HO-1 pET101,respectively (FIG. 12). An HPLC peak with an Rf value of 13.19 isobtained with the biliverdin commercial standard indicating that theblue-green material contains biliverdin in high concentration. Finally,mass spectral analyses of the purified blue-green material reveal apredominant molecular mass of 583.2-583.3 which is the expectedmonoisotopic mass of biliverdin IXα and that was also observed with thebiliverdin IXα commercial standard (FIG. 13). Altogether, the analyticalresults show that E. coli strains TOP10 or BL21 transformed with andexpressing HO1-pET101 or HemA-HO-1 pET101, respectively, producebiliverdin IXα when grown as described.

Example 5 Other Factors which Influence the Production of Biliverdin

The effect of trace metals on biliverdin production was tested. A tracemetal solution consisting of (per L) NaCl, 5 g; ZnSO₄-7H₂O, 1 g;MnCl₂-4H₂O, 4 g; FeCl₃-6H₂O, 4.75 g; CuSO₄-5H₂O, 0.4 g; H₃BO₃, 0.575 g;NaMoO₄-2H₂O, 0.5 g; and 6N H₂SO₄, ˜12.5 ml) was added to the growthmedium. The amount of biliverdin recovered with different amounts oftrace metal solution added is reported in Table 3.

TABLE 3 Effect of trace metal solution addition on biliverdin productiontrace metals solution Average amount BVIXα Range BVIXα (mL L⁻¹) produced(mg L⁻¹) produced (mg L⁻¹) 0 <0.1 <0.1 1.0 7.0  2.0-9.2 2.0 12  4.5-20

The concentration of dissolved oxygen during biliverdin production wasalso tested. dO₂ was measured by InPro® 6800 Series O₂ Sensors (METTLERTOLEDO) connected to BIOFLO® 310 Fermentation System (New BrunswickScientific). The results are plotted in FIG. 15. In general, thedissolved oxygen profile seen in FIG. 15 is associated with theproduction of biliverdin. In particular, the small spike (or peak) inoxygen concentration seen at about 5:07:40 is correlated with biliverdinproduction. Thus, if the dissolved oxygen concentration decreases fromits initial value to a first intermediate value, then increases to asecond intermediate value higher than the first intermediate value, thendecreases to a third intermediate value lower than the firstintermediate value, it is expected that the cells in the culture willproduce biliverdin.

Example 6 Biliverdin Production Using an Optimized PolynucleotideConstruct

The gene coding for HO1 was optimized to work in E. coli and synthesizedby DNA 2.0 Inc. (Menlo Park, Calif.). The coding sequence for a 6× Histag was incorporated at the 5′ end of the sequence so as to introducesix histidines to the N-terminus of HO1 expressed from the construct(FIG. 20). The synthesized HO1 gene was inserted into Vector pJexpress401 (DNA 2.0, Menlo Park, Calif.) to form vector HO1-pJ401 (FIG. 22).The vector HO1-pJ401 was transformed into E. coli BL21.

For biliverdin production, modified HO1-pJ401 containing E. coli strainswere grown in 80-ml of LB broth medium plus 100 μg per mL Kanamycin in250-mL capacity Erlenmeyer flasks in a shaker (225 rpm) at 37° C. to acell density showing an absorbance of 2 to 6 measured at 600 nm using a1 cm path length cuvette and LB broth medium as blank. The culture wasthen added to 2 L medium with 20 g NZamineA (Amersco, Solon Ohio), 10 gYeast extract (Fisher), 10 ml 100% Glycerol (Amersco, Solon Ohio), 20 mlLactose 20%, 5 ml Glucose 20%, 10 ml 200× Trace elements, 1 ml 2M MgSO₄,and 100 ml 20×NPS (Studier 2006) in NewBrunswick Bioflow 310 controllerused with bioCamand software for data collection, 40% dissolved oxygencascade control (0-30 percent O₂ supplemented to airflow, 280-500 rpmagitation, and 0.75-4 slpm airflow), and exponential feed, 200 ml 10%glycerol and 2% lactose feed. Blue-green pigmented material becamevisible 4 to 10 hours after lactose induction at or near the top of thefoam formed above the surface of the culture. Production of biliverdinincreased about two fold or more compared to the production in Example2.

The 200× trace elements solution was prepared by adding to a finalvolume of 250 ml: 0.5 ml HCl in 50 ml H₂O, FeCl₃ 0.675 g, CaCl₂ 0.15 g,MnCl₂ 0.1 g, ZnSO₄ 0.015 g, CoCl₂ 0.023 g, CuCl₂ 0.015 g, NiCl₂ 0.023 g,Na₂MoO₄ 0.025 g, and H₃BO₃ 0.007 g, then filter sterilizing. The 20×NPSstock solution was prepared by dissolving 66 g (NH₄)₂SO₄, 136 g KH₂PO₄,and 142 g Na₂HPO₄ in 1 L dd H₂O. The solution was autoclaved at 121° C.15 min.

It will be apparent to those having skill in the art that many changesmay be made to the details of the above-described embodiments withoutdeparting from the underlying principles of the invention. The scope ofthe present invention should, therefore, be determined only by thefollowing claims.

1. A non-animal cell comprising a recombinant heme oxygenase.
 2. Thenon-animal cell of claim 1, wherein the non-animal cell comprises avector comprising a polynucleotide that codes for the recombinant hemeoxygenase.
 3. The non-animal cell of claim 2, wherein the polynucleotidethat codes for the recombinant heme oxygenase comprises thepolynucleotide of SEQ ID NO:50.
 4. The non-animal cell of claim 1,wherein the non-animal cell comprises a regulatable promoter operablylinked to a polynucleotide that codes for the heme oxygenase.
 5. Thenon-animal cell of claim 1, wherein the heme oxygenase is a HO familyenzyme.
 6. The non-animal cell of claim 1, wherein the heme oxygenaseenzyme is HO1.
 7. The non-animal cell of claim 1, wherein the non-animalcell is an Escherichia coli cell.
 8. A method of producing biliverdinfrom a non-animal source, comprising: (a) culturing a non-animal cell ina growth medium, the cell comprising a recombinant heme oxygenase; and(b) isolating the biliverdin made in step (a).
 9. The method of claim 8,wherein the cell comprises a vector comprising a polynucleotide thatcodes for the recombinant heme oxygenase.
 10. The method of claim 8,wherein the cell comprises a regulatable promoter operably linked to apolynucleotide that codes for the heme oxygenase.
 11. The method ofclaim 9, wherein the polynucleotide that codes for the heme oxygenasecomprises the polynucleotide of SEQ ID NO:
 50. 12. The method of claim8, wherein the heme oxygenase is a HO family enzyme.
 13. The method ofclaim 8, wherein the heme oxygenase enzyme is HO1.
 14. The method ofclaim 8, wherein the non-animal cell is an Escherichia coli cell.
 15. Amethod of producing the non-animal cell of claim 1, comprisingintroducing into a parent non-animal cell a polynucleotide comprising asequence that codes for the recombinant heme oxygenase.
 16. The methodof claim 15, wherein the polynucleotide that codes for the recombinantheme oxygenase comprises the polynucleotide of SEQ ID NO:50.
 17. Themethod of claim 15, wherein the non-animal cell is an Escherichia colicell.